The dual-function phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is the second most frequently mutated gene in human cancers. PTEN counteracts the functions of many growth factors, the most prevalent of which is insulin-like growth factor II (IGF-II). PTEN expression is stimulated by IGF-II forming a feedback loop. Investigating IGF-binding protein (IGFBP) modulation of IGF-II actions on MCF-7 breast cancer cells, we found that IGFBP-2 also regulates PTEN. The MCF-7 cells were not responsive to high doses of IGF-II due to induction of PTEN, which was not observed with an IGF-II-analog that does not bind to IGFBPs or in the presence of an inhibitor that prevents IGFs associating with IGFBPs. These cells predominantly produce IGFBP-2: blocking IGFBP-2 with a specific antibody, or preventing IGFBP-2 binding to integrins, restored the induction of PTEN and the cells were non-responsive to high doses of the IGF-II-analog. Our findings indicate that breast cancer cells do not respond to high doses of IGF-II due to induction of PTEN, but IGFBP-2, when free from IGF-II can suppress PTEN. Levels of IGFBP-2 are elevated frequently in human tumors: its ability to regulate PTEN could have important implications in relation to therapeutic strategies targeting growth factor pathways.

Background: Breast density is positively associated with risk of breast cancer and the local microenvironment in the normal breast is known to significantly impact breast cancer initiation and further progression. The selective estrogen receptor modulator tamoxifen, with beneficial clinical effects on breast cancer recurrence, has also been shown to reduce mammographic density potentially explaining the primary preventive effects of tamoxifen. Still, important gaps persist regarding the cellular and molecular basis of how tamoxifen modifies breast density and subsequently breast cancer risk. Purpose: To investigate the impact by tamoxifen on normal human breast epithelial cells (MCF-10A) with the additional ambition to mimic variations in breast density on the cellular level. Experimental design: Effects of tamoxifen on MCF-10A cells at different cellular densities were evaluated in vitro. Flow cytometry and the sulforhodamine-B assay were used to assess cell cycle distribution and proliferation. Cell adhesion to extracellular matrix proteins was analyzed using hexosaminidase assay. Modulation of integrin receptor levels was determined by Western immunoblotting. Results: Tamoxifen exposure resulted in impaired cell cycle progression of human breast epithelial cells with a dose-dependent G2/M-phase arrest and reduced cell proliferation. Irrespective of tamoxifen treatment, the percent cellular adhesion to fibronectin and collagen type I by MCF-10A cells was significantly greater in high-density models compared to low-density models. Following tamoxifen treatment the adhesion was reduced in all density models. Consistent with the tamoxifen-induced lower adhesion, the integrin α1 and β3 levels were reduced in a dose-dependent way. Conclusion: These data support the hypothesis that tamoxifen affects the normal breast epithelium and its adhesion capacity, which may contribute to the clinically observed decrease in breast density and a potential reduced risk of breast tumor establishment. Citation Format: Rosendahl AH, Puig Blasco L, Borgquist S. Towards breast cancer prevention through reduced breast density: Suppressive effects of tamoxifen on normal breast epithelial cells [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-14-06.

Background: Treatment resistance is a well-known problem in estrogen receptor (ER) positive breast cancer. Complementary therapies are investigated for women who do not respond or who develop resistance against standard ER targeted treatment. Insulin-like growth factor-1 receptor (IGF1R) and its signaling pathway has been suggested to cause estrogen-independent cell growth and survival. Therapy against targets within the pathway is currently investigated in clinical trials. The aim of this study was to investigate if the IGF1R/mTOR pathway was activated or deregulated in breast cancer patients and to explore if any of the markers were prognostic, with or without adjuvant tamoxifen. Material and methods: Two patient cohorts were investigated by immunohistochemistry using tissue microarrays. The first cohort (N = 264) consisted of mainly post-menopausal women with stage II breast cancer treated with tamoxifen for 2 years irrespective of ER status. The second cohort (N = 206) consisted of mainly medically untreated, pre-menopausal patients with node-negative breast cancer. The protein expression of IGF1R, p-mTOR and p-S6rp were investigated. Cytoplasmic staining was evaluated for all markers and membrane staining was additionally evaluated for IGF1R. Statistical analyses were based on the intensity (0-3) of staining. Expression of IGF1R gave similar results in the cytoplasm and membrane, and p-values for cytoplasmic staining are reported below. Distant disease free survival (DDFS) at 5 years was used as end-point. Results: IGF1R expression was positively associated with ERa (p<0.001 in Mann-Whitney ranksum test), PgR (p<0.001) and HER2 (p = 0.042) expression in cohort 1, and also with Ki67 (p = 0.006) in cohort 2. p-S6rp was positively associated with ERα in cohort 1 (p<0.001) and HER2 (p = 0.004) in cohort 2. p-mTOR was positively associated only with Ki67 (p<0.001) in cohort 1. High expression of IGF1R was associated with a significantly better prognosis in cohort 1 (HR = 0.7 per intensity step, 95% CI = 0.5-0.9, p = 0.016 using Cox regression). When stratifying for ER status the effect was found in ER negative (ER-) (N = 80, HR = 0.6, 95% CI = 0.4-1.0, p = 0.03) but not in ER positive (ER+) patients (N = 174, HR 1.2, 95% CI = 0.8-2.0, p = 0.40). Both the effect in the ER- subgroup as well as the difference between ER- and ER+ patients were confirmed in interaction analysis and remains after adjustment for age, tumor size, node status, HER2, Ki67, and menopausal status (p = 0.06 for interaction). In cohort 2, no relation to DDFS could be found for IGF1R. p-mTOR and p-S6rp showed no relationship to prognosis in either of the cohorts. Conclusion: We found that high IGF1R expression was associated with a better prognosis for tamoxifen treated women. This effect could be seen in the ER- but not in the ER+ subgroup of patients. The lack of co-activation of downstream markers (p-mTOR and p-S6rp) in the IGF1R pathway shows that the prognostic effect is not due to complete activation of this pathway. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-52.